title: Glycosylation in a mammalian expression system is critical for the production of functionally active leukocyte immunoglobulin-like receptor A3 protein.
abstract: The leukocyte immunoglobulin-like receptor (LILR) A3 is a member of the highly homologous activating and inhibitory receptors expressed on leukocytes. LILRA3 is a soluble receptor of unknown functions but is predicted to act as a broad antagonist to other membrane-bound LILRs. Functions of LILRA3 are unclear primarily because of the lack of high quality functional recombinant protein and insufficient knowledge regarding its ligand(s). Here, we expressed and characterized recombinant LILRA3 (rLILRA3) proteins produced in 293T cells, Escherichia coli, and Pichia pastoris. We found that the purified rLILRA3 produced in the mammalian system was the same size as a 70-kDa native macrophage LILRA3. This is 20 kDa larger than the calculated size, suggesting significant post-translational modifications. In contrast, rLILRA3 produced in E. coli was similar in size to the unprocessed protein, but yeast-produced protein was 2-4 times larger than the unprocessed protein. Treatment with peptide-N-glycosidase F reduced the size of the mammalian cell- and yeast-produced rLILRA3 to 50 kDa, suggesting that most modifications are due to glycosylation. Consistent with this, mass spectrometric analysis of the mammalian rLILRA3 revealed canonical N-glycosylation at the predicted Asn(140), Asn(281), Asn(302), Asn(341), and Asn(431) sites. Functionally, only mammalian cell-expressed rLILRA3 bound onto the surface of monocytes with high affinity, and importantly, only this significantly abrogated LPS-induced TNFα production by monocytes. Binding to monocytes was partially blocked by β-lactose, indicating that optimally glycosylated LILRA3 might be critical for ligand binding and function. Overall, our data demonstrated for the first time that LILRA3 is a potential new anti-inflammatory protein, and optimal glycosylation is required for its functions.
authors.affiliation: From the Inflammation and Infection Research Centre, School of Medical Sciences.
authors.forename: Terry H Y
authors.initials: TH
authors.lastname: Lee
authors.forename: Ainslie
authors.initials: A
authors.lastname: Mitchell
authors.forename: Sydney
authors.initials: S
authors.lastname: Liu Lau
authors.forename: Hongyan
authors.initials: H
authors.lastname: An
authors.forename: Poornima
authors.initials: P
authors.lastname: Rajeaskariah
authors.forename: Valerie
authors.initials: V
authors.lastname: Wasinger
authors.forename: Mark
authors.initials: M
authors.lastname: Raftery
authors.forename: Katherine
authors.initials: K
authors.lastname: Bryant
authors.forename: Nicodemus
authors.initials: N
authors.lastname: Tedla
mesh_terms: D002460:Cell Line; D005260:Female; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D008297:Male; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011971:Receptors, Immunologic / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: C530062:LILRA3 protein, human; D011971:Receptors, Immunologic; D011994:Recombinant Proteins
keywords: Glycoprotein; Glycosylation; Leukocyte; Mass Spectrometry (MS); Monocytes; Protein Deamidation; Protein Expression; Protein Purification; Recombinant Protein Expression; Tumor Necrosis Factor (TNF)